Unmasking a two-faced protein

Single-molecule fluorescence spectroscopy and molecular dynamics simulations illuminate the structure and dynamics of PSD-95, a protein involved in neural plasticity.


NEUROTRANSMISSION
Unmasking a twofaced protein Single-molecule fluorescence spectroscopy and molecular dynamics simulations illuminate the structure and dynamics of PSD-95, a protein involved in neural plasticity.

IVAN MASLOV AND JELLE HENDRIX
T o help you process this article, billions of neurons in your brain have been learning for years how to convert images and letters on the screen into words and ideas. The biochemical background for learning and memory is neuroplasticity: neurons emerge and die, form new synapses, and abandon old ones. The strength of each connection is fine-tuned by protein molecules communicating with each other inside a synapse.
One of the most abundant proteins in the postsynaptic density -the area of the neuron where nerve signals are amplified or repressed -is a protein called postsynaptic density 95 (PSD-95). This protein modulates interactions between hundreds of other proteins ( Figure 1A; Wang et al., 2010;Fernández et al., 2009), and its structure and function change depending on synaptic activity (Bissen et al., 2019). Could it be that the different poses, or conformations, of the domains within PSD-95 specify which partners this protein interacts with inside the neuron, and hence allow neuroplasticity? Now, in eLife, a group led by Mark Bowen (from Stony Brook University), Feng Ding and Hugo Sanabria (both from Clemson University)with George Hamilton, Nabanita Saikia and Sujit Basak as joint first authors -reports on a structural model of PSD-95 based on single-molecule fluorescence microscopy and computer simulations. The group also confirms the predictions of the proposed model with a complimentary biochemical technique called disulfide screening, and reveal how ligand binding by one protein domain within PSD-95 is assisted by the proximity of another domain (Hamilton et al., 2022).
PSD-95 consists of five domains: three PDZ domains (PDZ1, PDZ2, PDZ3), an SH3 domain, and a GuK domain ( Figure 1B). Three of these domains -PDZ3, SH3, and GuK -associate closely with each other and form a conserved supramodule called PSG. The two other domains, PDZ1 and PDZ2, are separated from PSG by a long flexible linker, and mostly interact with each other rather than with the other domains (McCann et al., 2012;McCann et al., 2011). Within PSG, the interaction between domains SH3 and GuK is tight, while the PDZ3 domain can 'wiggle' around them (Korkin et al., 2006;Tavares et al., 2001). In computer simulations, the PDZ3 domain can reach both its closest neighbor, the SH3 domain, and the more distant GuK domain, but it remained unclear whether both or only one of the two PDZ3 conformations occur in nature ( Figure 1C; Korkin et al., 2006). Hamilton  showing that the PDZ3 domain switches between two major conformations with respect to the SH3-GuK complex ( Figure 1C). In particular, they used a technique called single-molecule Förster resonance energy transfer (smFRET) -frequently referred to as a 'molecular ruler' -to measure the distances between the domains in PSG. Combining these measurements with a simulation technique called 'rigid-body docking' revealed the atomic structures of both conformations.  Top: when observed at 10 frames per second (fps), the positions of the SH3 and GuK domains (which remain still) are correctly resolved, but the PDZ3 domain is switching between its positions in the two conformations of the PSG supramodule faster than the images are being taken. This results in an 'average' conformation being recorded (shown in blue) instead of the two 'real' conformations with the correct positions of the PDZ3 domain (red dashed circles). Center: when observed at 100 fps, a similar thing happens, but in this case two (incorrect) averaged conformations are observed. Bottom: 1000 fps is a high enough frame rate to correctly resolve the two conformations of the PSG supramodule. Observed averaged conformations are shown in blue and marked with an arrowhead; true underlying conformations are shown as red dashed circles. disulfide bonds with each other quicker when they are in close proximity. By substituting amino acid residues in PSD-95 for cysteines, Hamilton et al. were able to determine that residues that were presumed to be close together based on the proposed structures formed disulfide bonds faster than randomly selected pairs of residues.
Often, in realtime structural investigations like the ones performed by Hamilton et al., if a protein switches between two conformations faster than the detector can capture, the observed conformation will be a 'blurred' average, equally distant from the two extreme protein states ( Figure 1D). Previous experiments performed on the PSG supramodule used smFRET to capture slow protein dynamics, and were only able to capture a single structure for the module (McCann et al., 2012). This result was inconsistent with structures for PSD-95 determined using other biophysical methods, such as SAXS or NMR (Zhang et al., 2013).
The observation by   for further intriguing investigations. For example, how are the conformations and structural dynamics of PSD-95 affected by ligands, protein partners, or post-translational modifications? It will also be important to determine whether there are protein partners and ligands that preferentially bind PSD-95 in the conformation in which the PDZ3 domain directly interacts with the Guk domain? This is, definitely, something for us and the neurons in our brains to learn in the future.